Transplantation of Testis Stem Cells in Large Animals The testis stem cell is unique among the stem cells in an adult male body in that it is the only cell that divides and can contribute genes to subsequent generations. Donor-derived spermatogenesis in a recipient testis after transplantation provides the only unequivocal bioassay system for functional competence of spermatogonial stem cells and is a powerful approach for the study of spermatogenesis. To date, germ cell transplantation has been performed predominantly in rodents. The long-term objective of the proposed research is to establish germ cell transplantation in a non-rodent mammalian species as a unique tool to study the biology of germ line stem cells, serve as a bioassay for stem cell potential, and allow the manipulation of different aspects of spermatogenesis. Pigs will be used as a non-rodent model species because they are readily obtained, relatively inexpensive to maintain and mature quickly. Due to their size and testicular anatomy, the results obtained in pigs will be applicable to other large animal species and man. The project will address 1) the selection of donor cells for germ line stem cells, 2) the introduction of a genetic marker to follow the progression of donor spermatogenesis in the recipient testis and to lay the foundation for modification of the male germ line, 3) the modification of the recipient testis to suppress endogenous spermatogenesis and to provide a permissive environment for donor-derived spermatogenesis, and 4) grafting of donor testis tissue under the back skin of mice as a novel in vivo culture system for the study of spermatogenesis and as an alternate approach to transplantation of isolated germ cells. This system will lay the foundation for modification of the male germ line as an alternate approach to generate germ line transgenic animals, will serve to preserve valuable genetic material and can be clinically applicable to restore fertility in patients undergoing cytotoxic therapy for cancer. The results of this project are expected to provide a novel approach for in depth investigations into germ line stem cell biology and male fertility in non-rodent mammalian species.